G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae (original) (raw)
Summary
Coding sequence cartridges for aminoglycoside phosphotransferase (APT) were isolated from bacterial transposon Tn903. When incorporated into a heterologous gene construction utilising the PGK1 promoter and terminator, the heterologous APT gene provided a G418-resistance determinant that functioned efficiently as a dominant marker for yeast in both multiple- and single-copy. Transformant colonies on selective medium appeared rapidly, within 36–48 h, and growth rate of the transformed cells was normal. A simple and highly sensitive radiolabelling assay for APT enzyme activity was developed for use with crude cell protein extracts. Enzyme activity units were equated to the amount of APT protein present in the cells, and the APT protein was shown to be stable in yeast. Heterologous APT expression was 130-fold reduced compared with homologous PGK1. This resulted from an estimated two-fold decrease in mRNA level and a 65-fold decrease in translation efficiency. The latter was unaffected by AUG sequence context change, but corresponded with a high frequency of minor codons in the _APT_-coding sequence. APT can be used as a semi-quantitative reporter of gene expression, whose useful features are in vivo detection via the G418-resistance phenotype and powerful cell-free assay.
Access this article
Subscribe and save
- Get 10 units per month
- Download Article/Chapter or eBook
- 1 Unit = 1 Article or 1 Chapter
- Cancel anytime Subscribe now
Buy Now
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Instant access to the full article PDF.
Similar content being viewed by others
References
- Baim SB, Sherman F (1988) Mol Cell Biol 8:1591–1602
Google Scholar - Bennetzen JL, Hall BD (1982) J Biol Chem 257:3026–3031
Google Scholar - Boulnois GJ (ed) (1987) Gene cloning and analysis: a laboratory guide. Blackwell Scientific Publications, Oxford, UK
Google Scholar - Bradford MM (1976) Anal Biochem 147:248–254
Google Scholar - Casey GP, Xiao W, Rank GH (1988) J Inst Brew 94:93–97
Google Scholar - Cashmore AM, albury MS, Hadfield C, Meacock PA (1986) Mol Gen Genet 203:154–162
Google Scholar - chen CY, Oppermann H, Hitzeman RA (1984) Nucleic Acid Res 12:8951–8970
Google Scholar - Cigan AM, Donahue TF (1987) Gene 59:1–18
Google Scholar - Fogel S, Welch J (1982) Proc Natl Acad Sci USA 79:5342–5346
Google Scholar - Gale EF, Cundliffe E, Reynolds PE, Richmond MH, Waring MJ (1981) The Molecular Basis of Antibiotic Action, 2nd, edn. Wiley, London, pp 503–506
Google Scholar - Haas MJ, Dowding JE (1975) Methods Enzymol 43:611–628
Google Scholar - Hadfield C, Cashmore AM, Meacock PA (1986) Gene 45:149–158
Google Scholar - Hadfield C, Cashmore AM, Meacock PA (1987) Gene 52:59–70
Google Scholar - Henderson RCA, Cox BS, Tubb R (1985) Curr Genet 9:133–138
Google Scholar - Hames BD (1981) An introduction to polyacrylamide gel electrophoresis. In: Hames BD, Rickwood D (eds) Gel electrophoresis of proteins. IRL Press, Oxford, pp 1–91
Google Scholar - Hoekema A, Kastelein RA, Vasser M, deBoer HA (1987) Mol Cell Biol 7:2914–2924
Google Scholar - Hollenberg CP (1982) Curr Topics Microbiol Immunol 96:119–144
Google Scholar - Ito H, Fukuda Y, Murata K, Kimra A (1983) J Bacteriol 153:163–168
Google Scholar - Jimenez A, Davies J (1980) Nature 287:869–871
Google Scholar - Kozak M (1986) Cell 44:283–292
Google Scholar - Kozak M (1989) Mol Cell Biol 9:5134–5142
Google Scholar - Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Google Scholar - Mellor J, Dobson MJ, Roberts NA, Kingsman AJ, Kingsman SM (1985) Gene 33:215–226
Google Scholar - Mellor J, Dobson MJ, Kingsman AJ, Kingsman SM (1987) Nucleic Acids Res 15:6243–6260
Google Scholar - Norrander J, Kempe T, Messing J (1983) Gene 26:101–106
Google Scholar - Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Proc Natl Acad Sci USA 78:6354–6358
Google Scholar - Parent SA, Fenimoe CM, Bostian KA (1985) Yeast 1:83–138
Google Scholar - Reiss B, Sprengel R, Will H, Schaller H (1984) Gene 30:211–218
Google Scholar - Sharp PM, Tuohy TMF, Mosurski KR (1986) Nucleic Acids Res 14:5125–5143
Google Scholar - Sherman F, Stewart JW (1982) Mutations altering initiation of translation of yeast iso-1-cytochrome c; contrasts between the eukaryotic and prokaryotic initiation process. In: Strathern NJ, Jones EW, Broach JR (ed) The molecular biology of the yeast Saccharomyces. Metabolism and gene expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 301–333
Google Scholar - Shine J, Dalgarno L (1974) Proc Natl Acad Sci USA 71:1342–1346
Google Scholar - Stark MJR (1987) Gene 51:255–267
Google Scholar - Vieira J, Messing J (1982) Gene 19:259–268
Google Scholar - Webster TD, Dickson RC (1983) Gene 26:243–252
Google Scholar - Zhu J, Contreras R, Gheysen D, Erst J, Fiers W (1985) Biotechnology 3:451–456
Google Scholar - Zitomer RS, Hall D (1976) J Biol Chem 251:6320–6326
Google Scholar - Zitomer RS, Walthall DA, Rymond BC, Hollenberg CP (1984) Mol Cell Biol 4:1191–1197
Google Scholar
Author information
Authors and Affiliations
- Leicester Biocentre, University of Leicester, LE1 7RH, Leicester, England, UK
C. Hadfield, B. E. Jordan, R. C. Mount, G. H. J. Pretorius & E. Burak
Authors
- C. Hadfield
You can also search for this author inPubMed Google Scholar - B. E. Jordan
You can also search for this author inPubMed Google Scholar - R. C. Mount
You can also search for this author inPubMed Google Scholar - G. H. J. Pretorius
You can also search for this author inPubMed Google Scholar - E. Burak
You can also search for this author inPubMed Google Scholar
Rights and permissions
About this article
Cite this article
Hadfield, C., Jordan, B.E., Mount, R.C. et al. G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae.Curr Genet 18, 303–313 (1990). https://doi.org/10.1007/BF00318211
- Received: 01 July 1990
- Issue Date: November 1990
- DOI: https://doi.org/10.1007/BF00318211